Cellular ATP levels alone do not reliably reflect overall mitochondrial bioenergetics or mitochondrial dysfunction in Barth syndrome

Abstract

This review critically examines the question posed by Brault JF, Simon J, and Conway SJ in Journal of Translational Genetics and Genomics earlier this year: “What can ATP content tell us about Barth syndrome muscle phenotypes?”. It also offers an alternative perspective on the topic. Our answer is straightforward but warrants a detailed explanation. In the early stages of Barth syndrome, measuring adenosine triphosphate (ATP) content alone is insufficient to accurately characterize the bioenergetic profile of cells or tissues. Nevertheless, such measurements continue to attract interest - even from researchers equipped with advanced techniques - including adenosine diphosphate (ADP) and ATP assays, adenosine monophosphate (AMP) quantification, and assessments of nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide Adenine dinucleotide (NADH) [i.e., nicotinamide adenine dinucleotide phosphate oxidized (NADP⁺) or nicotinamide adenine dinucleotide oxidized (NAD⁺)]. Assessing the rate of ATP production is a more informative and complementary approach, offering greater insight than ATP measurements alone. However, adopting a bioenergetic framework for studying Barth syndrome remains challenging. This difficulty arises largely from the profound structural and functional changes occurring within the mitochondrial compartment, which affect not only transmembrane ion transport but also the import and maturation of cytoplasmic precursors of mitochondrial proteins. Future research on Barth syndrome (BTHS) is expected to shift focus toward the central role of immature cardiolipin and monolysocardiolipin in mitochondrial membranes and their complex interactions, rather than concentrating solely on disruptions in cellular bioenergetics.

Keywords

ATP determination / Barth syndrome / bioenergetics / cardiolipin / mitochondria / monolysocardiolipin

References

[1] DudekJ.Barth syndrome cardiomyopathy.Cardiovasc Res2017;113:399-410

[2] ChathamJC.Metabolic remodeling in the hypertrophic heart: fuel for thought.Circ Res2012;111:666-8 PMCID:PMC3462817

[3] NickelA,MaackC.Myocardial energetics in heart failure.Basic Res Cardiol2013;108:358

[4] BraultJJ.What can ATP content tell us about Barth syndrome muscle phenotypes?.J Transl Genet Genom2025;9:1-10 PMCID:PMC11951242

[5] BarthPG,BerdenJA.An X-linked mitochondrial disease affecting cardiac muscle, skeletal muscle and neutrophil leucocytes.J Neurol Sci1983;62:327-55

[6] BrandMD.Assessing mitochondrial dysfunction in cells.Biochem J2011;435:297-312 PMCID:PMC3076726

[7] WuM,SwiftAL.Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.Am J Physiol Cell Physiol2007;292:C125-36

[8] YuristaSR,RosenzweigA,WestenbrinkBD.Ketone bodies for the failing heart: fuels that can fix the engine?.Trends Endocrinol Metab2021;32:814-26

[9] SaricA,ArmandAS,PetitPX.Barth syndrome: from mitochondrial dysfunctions associated with aberrant production of reactive oxygen species to pluripotent stem cell studies.Front Genet2015;6:359 PMCID:PMC4719219

[10] NeusteinHB,DahmsB.An X-linked recessive cardiomyopathy with abnormal mitochondria.Pediatrics1979;64:24-9

[11] BarthPG,BowenVM.X-linked cardioskeletal myopathy and neutropenia (Barth syndrome): an update.Am J Med Genet A2004;126A:349-54

[12] GonzalvezF,DupaigneP.tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak.Cell Death Differ2005;12:614-26

[13] BazánS,MallampalliVK,SparagnaGC.Cardiolipin-dependent reconstitution of respiratory supercomplexes from purified Saccharomyces cerevisiae complexes III and IV.J Biol Chem2013;288:401-11

[14] PfeifferK,StuartRA.Cardiolipin stabilizes respiratory chain supercomplexes.J Biol Chem2003;278:52873-80

[15] GonzalvezF,BoutantM.Barth syndrome: cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation.Biochim Biophys Acta2013;1832:1194-206

[16] GonzalvezF,HoutkooperRH.Cardiolipin provides an essential activating platform for caspase-8 on mitochondria.J Cell Biol2008;183:681-96 PMCID:PMC2582890

[17] Saini-ChohanHK,ChiccoAJ.Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.J Lipid Res2009;50:1600-8

[18] KhuchuaZ,BattsL.A zebrafish model of human Barth syndrome reveals the essential role of tafazzin in cardiac development and function.Circ Res2006;99:201-8

[19] LiXX,LiYF,HeRR.Cardiolipin and its different properties in mitophagy and apoptosis.J Histochem Cytochem2015;63:301-11 PMCID:PMC4409943

[20] GawrischK.Tafazzin senses curvature.Nat Chem Biol2012;8:811-12 PMCID:PMC3478944

[21] GebertN,KutikS.Mitochondrial cardiolipin involved in outer-membrane protein biogenesis: implications for Barth syndrome.Curr Biol2009;19:2133-9 PMCID:PMC4329980

[22] JiangF,SchlameM.Absence of cardiolipin in the crd1 null mutant results in decreased mitochondrial membrane potential and reduced mitochondrial function.J Biol Chem2000;275:22387-94

[23] ZhangM,DowhanW.Gluing the respiratory chain together: Cardiolipin is required for supercomplex formation in the inner mitochondrial membrane.J Biol Chem2002;277:43553-6

[24] BanT,SongZ,ChanDC.OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation.Hum Mol Genet2010;19:2113-22 PMCID:PMC2865371

[25] DeVayRM,LacknerLL,StahlbergH.Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion.J Cell Biol2009;186:793-803 PMCID:PMC2753158

[26] JoshiAS,FeiN,GreenbergML.Cardiolipin and mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in Saccharomyces cerevisiae.J Biol Chem2012;287:17589-97 PMCID:PMC3366806

[27] PatilVA.Cardiolipin-mediated cellular signaling. In: Capelluto DG, editor. Lipid-mediated protein signaling. Dordrecht: Springer Netherlands; 2013. pp. 195-213.

[28] IkonN,HsuFF,RyanRO.Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.Biochem Biophys Res Commun2015;464:580-5 PMCID:PMC4522224

[29] HeitB,GrinsteinS.Changes in mitochondrial surface charge mediate recruitment of signaling molecules during apoptosis.Am J Physiol Cell Physiol2011;300:C33-41

[30] KimTH,DingWX.Bid-cardiolipin interaction at mitochondrial contact site contributes to mitochondrial cristae reorganization and cytochrome C release.Mol Biol Cell2004;15:3061-72

[31] ManganelliV,RecalchiS.Altered Traffic of cardiolipin during apoptosis: exposure on the cell surface as a trigger for “antiphospholipid antibodies”.J Immunol Res2015;2015:847985 PMCID:PMC4603604

[32] ChuCT,KaganVE.LC3 binds externalized cardiolipin on injured mitochondria to signal mitophagy in neurons: implications for Parkinson disease.Autophagy2014;10:376-8 PMCID:PMC5396091

[33] GuZ,ChenS.Aberrant cardiolipin metabolism in the yeast taz1 mutant: a model for Barth syndrome.Mol Microbiol2004;51:149-58

[34] SchlameM,GreenbergML.The biosynthesis and functional role of cardiolipin.Prog Lipid Res2000;39:257-88

[35] KoshkinV.Oxidative phosphorylation in cardiolipin-lacking yeast mitochondria.Biochem J2000;347:687-91 PMCID:PMC1221004

[36] KadenbachB,KolbeHV,PalmieriF.The mitochondrial phosphate carrier has an essential requirement for cardiolipin.FEBS Lett1982;139:109-12

[37] RobinsonNC.Functional binding of cardiolipin to cytochrome c oxidase.J Bioenerg Biomembr1993;25:153-63

[38] NoëlH.An essential requirement of cardiolipin for mitochondrial carnitine acylcarnitine translocase activity. Lipid requirement of carnitine acylcarnitine translocase.Eur J Biochem1986;155:99-102

[39] VazFM,ValianpourF,WandersRJ.Only one splice variant of the human TAZ gene encodes a functional protein with a role in cardiolipin metabolism.J Biol Chem2003;278:43089-94

[40] BrandnerK,FrazierAE,MeisingerC.Taz1, an outer mitochondrial membrane protein, affects stability and assembly of inner membrane protein complexes: implications for Barth Syndrome.Mol Biol Cell2005;16:5202-14 PMCID:PMC1266419

[41] XuY,BlanckTJ.Remodeling of cardiolipin by phospholipid transacylation.J Biol Chem2003;278:51380-5

[42] AcehanD,StokesDL.Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography.Lab Invest2007;87:40-8 PMCID:PMC2215767

[43] XuY,RenM.The enzymatic function of tafazzin.J Biol Chem2006;281:39217-24

[44] McKenzieM,ThorburnDR.Mitochondrial respiratory chain supercomplexes are destabilized in Barth syndrome patients.J Mol Biol2006;361:462-9

[45] XuY,PleskenH,SchlameM.Characterization of lymphoblast mitochondria from patients with Barth syndrome.Lab Invest2005;85:823-30

[46] XuY,PleskenH.A Drosophila model of Barth syndrome.Proc Natl Acad Sci U S A2006;103:11584-8

[47] PhoonCK,SchlameM.Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction.J Am Heart Assoc2012;1

[48] AcehanD,HoutkooperRH.Cardiac and skeletal muscle defects in a mouse model of human Barth syndrome.J Biol Chem2011;286:899-908

[49] ZhuS,ZhuM.Cardiolipin remodeling defects impair mitochondrial architecture and function in a murine model of Barth syndrome cardiomyopathy.Circ Heart Fail2021;14:e008289

[50] WangG,YangL.Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies.Nat Med2014;20:616-23

[51] HeQ.Tafazzin knockdown causes hypertrophy of neonatal ventricular myocytes.Am J Physiol Heart Circ Physiol2010;299:H210-6

[52] DesousaBR,JonesAE.Calculation of ATP production rates using the seahorse XF analyzer.EMBO Rep2023;24:e56380 PMCID:PMC10561364

[53] DoerrierC,KrumschnabelG,MészárosAT.High-resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. In: Palmeira CM, Moreno AJ, editors. Mitochondrial bioenergetics. New York: Springer; 2018. pp. 31-70.

[54] GuX,LiuY.Measurement of mitochondrial respiration in adherent cells by seahorse XF96 cell mito stress test.STAR Protoc2021;2:100245 PMCID:PMC7797920

[55] MookerjeeSA,NichollsDG.Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements.J Biol Chem2017;292:7189-207 PMCID:PMC5409486

[56] PelletierM,RamaswamyM.Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption.Methods Enzymol2014;542:125-49.

[57] DivakaruniAS.A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements.Nat Metab2022;4:978-94 PMCID:PMC9618452

[58] MillerSG,BraultJJ.Increased adenine nucleotide degradation in skeletal muscle atrophy.Int J Mol Sci2019;21:88 PMCID:PMC6981514

[59] deKok MJC,WüstRCI.Circumventing the crabtree effect in cell culture: a systematic review.Mitochondrion2021;59:83-95

[60] HandelME,MookerjeeSA.The whys and hows of calculating total cellular ATP production rate.Trends Endocrinol Metab2019;30:412-6

[61] TullsonPC,TerjungRL.Adenine nucleotide degradation in slow-twitch red muscle.Am J Physiol1990;258:C258-65

[62] LawAS,BraultJJ.Liquid chromatography method for simultaneous quantification of ATP and its degradation products compatible with both UV-Vis and mass spectrometry.J Chromatogr B Analyt Technol Biomed Life Sci2022;1206:123351 PMCID:PMC9479163

[63] PetitPX,SchrantzN.Oxidation of pyridine nucleotides during Fas- and ceramide-induced apoptosis in Jurkat cells: correlation with changes in mitochondria, glutathione depletion, intracellular acidification and caspase 3 activation.Biochem J2001;353:357-67

[64] HancockCR,WisemanRW,MeyerRA.31P-NMR observation of free ADP during fatiguing, repetitive contractions of murine skeletal muscle lacking AK1.Am J Physiol Cell Physiol2005;288:C1298-304

[65] MarvinJS,KumarM.iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.Proc Natl Acad Sci U S A2024;121:e2314604121 PMCID:PMC11126915

[66] StanleyPE.Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme.Anal Biochem1969;29:381-92

[67] YangNC,ChenYH.A convenient one-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP.Anal Biochem2002;306:323-7

[68] TullsonPC.Adenine nucleotide degradation in striated muscle.Int J Sports Med1990;11 Suppl 2:S47-55

[69] deTaffin de Tilques M,GodardF.Decreasing cytosolic translation is beneficial to yeast and human Tafazzin-deficient cells.Microb Cell2018;5:220-32 PMCID:PMC5961916

[70] WangX.A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death.Nature2015;524:481-4 PMCID:PMC4582408

[71] DuncanAL.Monolysocardiolipin (MLCL) interactions with mitochondrial membrane proteins.Biochem Soc Trans2020;48:993-1004 PMCID:PMC7329354